Abstract
The Exon/Intron Database (ExInt) stores information of all GenBank eukaryotic entries containing an annotated intron sequence. Data are available through a retrieval system, as flat-files and as a MySQL dump file. In this report we discuss several implementations added to ExInt, which is accessible at http://intron.bic.nus.edu.sg/exint/newexint/exint.html.
INTRODUCTION
The exponential growth of sequence databases, especially due to genome and EST sequencing, has generated a parallel increase in the amount of sequences showing an intron/exon organization. We have recently developed a database containing all sequences in GenBank bearing in their annotation at least one exon/intron boundary (1). This, and other related databases (2,3), has been used in several studies approaching issues related to the exon/intron organization of eukaryotic genes (4,5).
In this report, we describe a series of implementations to the Exon/Intron Database (ExInt) as follows:
1. Relational database: data are now stored in a relational database (MySQL). The table structure is presented in Figure 1. Data from the database tables can be downloaded in a dump format, which allows direct incorporation in other MySQL relational databases.
Figure 1.
Description of the ExInt relational database.
2. Purged database: it is known that GenBank is extremely redundant. To avoid any potential bias, we have made available in this latest version of ExInt a non-redundant set of the data. Overall analysis of both redundant and non-redundant sets confirmed that most of the sequences (>80%) are redundant in current databases. Both datasets are available for download as Fasta libraries. They are also searchable using ExInt Blast engine.
3. Statistics link: several statistical features (for the whole database and models species) are available, such as number of genes, exons and introns before and after purging (Table 1); exon length distribution (Fig. 2); intron length distribution (Fig. 3) and intron phase distribution (Table 2).
Table 1. Gene, exon and intron number for whole ExInt and subdivisions.
| Gene number | Exon number | Intron number | |
|---|---|---|---|
| Whole ExInt | 94 615 | 518 169 | 525 870 |
| Non-redundant ExInt | 15 271 | 113 457 | 128 065 |
| Rattus norvegicus | 835 | 4889 | 7191 |
| Homo sapiens | 8287 | 60 499 | 43 127 |
| Mus musculus | 3044 | 18 920 | 15 407 |
| Drosophila melanogaster | 15 220 | 64 271 | 89 969 |
| Caenorhabditis elegans | 18 924 | 121 708 | 108 803 |
| Arabidopsis thaliana | 25 216 | 158 629 | 127 386 |
| Saccharomyces cerevisiae | 589 | 1695 | 1438 |
Figure 2.
Exon size distribution. The complete database is shown in black, a non-redundant set is shown in red.
Figure 3.
Intron size distribution. The complete database is shown in black, a non-redundant set is shown in red. The yellow line corresponds to experimentally defined introns.
Table 2. Intron phase distribution.
| 0 | 1 | 2 | |
|---|---|---|---|
| All ExInt | 257 713 (49%) | 147 625 (28%) | 120 532 (23%) |
| Non-redundant | 60 979 (48%) | 35 438 (28%) | 31 608 (24%) |
| Rattus norvegicus | 2842 (39%) | 2365 (33%) | 1384 (28%) |
| Mus musculus | 6703 (44%) | 5921 (38%) | 2783 (18%) |
| Caenorhabditis elegans | 51 251 (47%) | 28 553 (26%) | 28 999 (27%) |
| Homo sapiens | 19 102 (44%) | 15 423 (36%) | 8602 (20%) |
| Arabidopsis thaliana | 71 958 (56%) | 28 178 (22%) | 27 250 (22%) |
| Drosophila melanogaster | 38 101 (42%) | 28 896 (32%) | 22 972 (26%) |
| Saccharomyces cerevisiae | 641 (45%) | 428 (30%) | 369 (25%) |
4. Validation of predicted gene structure using EST data: we provided a validated subset for genes predicted in seven species: Homo sapiens, Mus musculus, Rattus sp., Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Saccharomyces cerevisae (Table 3).
Table 3. Predicted introns confirmed by EST.
| GenBank ID with predicted introns | GenBank ID with confirmed predicted introns | Predicted introns | Number of ESTs | Predicted introns confirmed by ESTs | |
|---|---|---|---|---|---|
| Rattus norvegicus | 23 | 10 | 183 | 273591 | 31 (17%) |
| Mus musculus | 137 | 73 | 1704 | 1 29633 2 | 389 (23%) |
| Caenorhabditis elegans | 3016 | 2283 | 100 977 | 58 367 | 17454 (17%) |
| Homo sapiens | 1852 | 1149 | 23 235 | 340 6430 | 6013 (26%) |
| Arabidopsis thaliana | 1592 | 1438 | 125 567 | 112 999 | 31873 (25%) |
| Drosophila melanogaster | 703 | 542 | 52 639 | 116 099 | 10278 (20%) |
| Saccharomyces cerevisiae | 317 | 38 | 1024 | 11 159 | 38 (4%) |
METHODOLOGY
We have used GenBank release 122 to construct a raw database containing all eukaryotic sequences with an exon/intron organization. The approach used to identify all intron-containing sequences in GenBank has been described previously (1). The same is true for the methodology used to construct the following derived databases: predicted introns, experimentally defined introns, organelle and nuclear genes (1). A purged database was constructed using a modification of the method of Long et al. (6), as follows. We performed an all-against-all protein sequence comparison using a PVM-version of Fasta in an eight-node cluster of PCs running Linux. When two protein sequences have an identity level ≥25% over at least 70% of the length of the shorter sequence, just one sequence is kept. These comparisons are exhaustive until a complete non-redundant database is obtained. As a representative of the gene cluster we have taken the sequence with the largest number of exons and introns.
To validate the predicted gene structures, we take the predicted cDNA structure (keeping the positional information of all predicted introns) for all genes within seven model species and used Blast (7) to search them against the respective (same species) EST datasets. A script in PERL was written to parse the Blast output looking for cases where a predicted exon/exon boundary (by that we mean a region in the cDNA where a predicted intron is present at the genomic level) was confirmed by at least one EST.
RESULTS AND DISCUSSION
ExInt contains a wealth of relevant biological information. Here, we present some statistics that are important to the database construction and for a general evaluation of the data. Table 1 shows the number of genes, exons and introns for the redundant and non-redundant datasets and for seven model species. We note that there are, on average, 5.48 exons per gene with AL445795 having the higher number (96). Figures 2 and 3 show the exon and intron length distributions, respectively. We confirm an observation from Deutsch and Long (8) that invertebrate introns are on average smaller than human introns. As also seen by Deutsch and Long (8), we have observed a bimodal distribution of intron length for the whole dataset, which does not seem to be due to predicted introns, since the same pattern is also observed for the confirmed introns (Fig. 3). Positioning of introns along the coding region (Fig. 4) shows a bias distribution towards the C-terminal half of the protein molecule. This piece of information is important for interpretation of data related to gene structure. For example, it has recently been suggested that alternative splicing events are more frequent on the C-terminal half of proteins (9), a bias that can be due to the distribution shown in Figure 4.
Figure 4.
Intron phase distribuition along the cds. Black, introns phase 0; red, introns phase 1; yellow, introns phase 2.
The validation of predicted gene structures is probably the most important implementation to ExInt. It has been shown that gene prediction programs may generate a large amount of artefactual gene structures, and analysis using these datasets may draw incorrect conclusions (10). We have made use of the large amount of EST data available in dbEST to validate the predicted gene structure for sequences of seven different model species, H.sapiens, M.musculus, Rattus sp., C.elegans, D.melanogaster, A.thaliana and S.cerevisae. This validation step creates a sub-set of ‘trusted’ predicted gene structure that may be important in a number of biological queries. The sub-set of validated intron/exon boundaries may also constitute a useful resource for developers of gene prediction programs. It is important to emphasize that the absence of validation does not imply that the predicted gene structure is wrong, since the coverage of the transcriptome by ESTs is not yet complete.
AVAILABILITY
ExInt is accessible via a World Wide Web interface at http://intron.bic.nus.edu.sg/exint/newexint/exint.html. Different features can be used as a query element such as: NID, locus name and keyword. The whole database, as well as derived databases, is available for download. Derived databases include: purged database, predicted intron, experimentally defined introns, organelle genes and nuclear genes. Users can also search all databases with a query sequence using Blast. ExInt will be updated twice a year.
ACKNOWEDGEMENT
F.P. is supported by Fapesp (00/02228-9).
Table 4. Frequency of exon symmetry.
| 0.0 | 0.1 + 1.0 | 1.1 | 1.2 +2.1 | 2.2 | 0.2 + 2.0 | |
|---|---|---|---|---|---|---|
| Whole ExInt | 111 959 | 97 398 | 37 923 | 50 644 | 24 475 | 92 348 |
| Non-redundant | 26 878 | 25 491 | 9729 | 14 004 | 7290 | 24 803 |
| Rattus norvegicus | 497 | 448 | 1474 | 1017 | 62 | 1855 |
| Mus musculus | 3037 | 3037 | 2264 | 1547 | 470 | 2189 |
| Caenorhabditis elegans | 20 422 | 20 814 | 7009 | 11 898 | 7022 | 21 448 |
| Homo sapiens | 8552 | 8989 | 5289 | 5072 | 1645 | 6581 |
| Arabidopsis thaliana | 35 951 | 22 991 | 5623 | 9216 | 5245 | 24 420 |
| Drosophila melanogaster | 11 898 | 15 756 | 6821 | 10 083 | 4967 | 13 691 |
| Saccharomyces cerevisiae | 99 | 84 | 27 | 79 | 24 | 86 |
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